This proposal outlines a research effort centered on three important folate requiring enzymes: two involved in de novo purine biosynthesis, the human and avian bifunctional 5'-aminoimidazole-4 carboxamide ribonucleotide transformylase (AICARTfase) inosine monophosphate cyclohydrolase, the mono- (E.coli) and trifunctional (human) proteins possessing glycinamide ribonucleotide transformylase (GARTfase) activity, and the third, dihydrofolate reductase from E.coli and human sources. Experiments are designed to: 1) elaborate the minimal kinetic schemes through pretransient and steady state kinetics for formyl transfer by the two transformylase enzymes in order to provide a basis for: studies with mutants aimed at implicating a putative proton shuttle mechanism in the action of GARTfase, assessing the effect of neighboring domains on the kinetic properties of a GARTfase active site imbedded in the polyfunctional human form of the protein, and establishing a kinetic and mechanistic identity between the GARTfase and AICARTfase enzymes despite an absence of sequence homology; 2) Gain evidence for a link between the dynamic fluctuations in the protein structure of dihydrofolate reductase as monitored by 3D NMR and its catalytic activity; and 3) seek data for channeling in a putative de novo purine biosynthetic complex through the technique of confocal fluorescent microscopy using fluorescently tagged native and mutant forms of human GARTfase and AICARTfase. These folate requiring enzymes are important in one carbon unit transfer and have been the target of chemotherapy. Dihydrofolate reductase in particular serves as a paradigm for understanding the fundamentals of enzyme catalysis.